Assessing serum levels of SM22α as a new biomarker for patients with aortic aneurysm/dissection

Background Aortic aneurysm/dissection (AAD) is now encountered more often because of the increasing prevalence of atherosclerosis and hypertension in the population. Despite many therapeutic improvements, in particular timely and successful surgery, in-hospital mortality rates are still higher. Timely identification of patients at high risk will help improve the overall prognosis of AAD. Since early clinical and radiological signs are nonspecific, there is an urgent need for accurate biomarkers. Smooth muscle 22α (SM22α) is a potential marker for AAD because of its abundant expression in vascular smooth muscle, which is involved in development of AAD. Methods We prepared three different mouse models, including abdominal aortic aneurysm, neointimal hyperplasia and atherosclerosis. SM22α levels were assessed in serum and vascular tissue of the mice. Next, the relationships between serum SM22α level and vascular lesion were studied in mice. Finally, serum from 41 patients with AAD, 107 carotid artery stenosis (CAS) patients and 40 healthy volunteers were tested for SM22α. Serum levels of SM22α were measured using an enzyme-linked immunosorbent assay (ELISA). Results Compared with the controls, serum SM22α levels were reduced in the models of aortic aneurysm, neointimal formation and atherosclerosis, and elevated in mice with ruptured aneurysm. Serum SM22α level was negatively correlated with apoptosis rate of vascular smooth muscle cells (VSMC), ratio of intima/ media (I/M) area and plaque size. Patients with AAD had significantly higher serum SM22α levels than patients with only CAS, or normal controls. Conclusion Serum SM22α could be a potential predictive marker for AAD, and regulation of VSMC is a possible mechanism for the effects of SM22α.


Introduction
Aortic aneurysm/dissection (AAD) is one of the most life-threatening disease, associated with high rates of mortality in case of aortic rupture [1,2]. For all patients with AAD, the early diagnosis and treatment is crucial for improved survival. However, due to the lack of typical clinical symptoms, AAD is often misdiagnosed as angina, acute myocardial infarction or other cardiovascular diseases, resulting in the delay of treatment. Currently, the diagnosis and follow-up of AAD mainly depends on various imaging techniques, such as magnetic resonance angiography, computed tomography angiography, or ultrasound, which are relatively timeconsuming in some emergency situations. Therefore, blood-derived biomarkers may be more suitable for the rapid diagnosis of AAD.
The structural and functional changes in the vascular smooth muscle cells (VSMC) including phenotypic transformation and apoptosis play a critical role in the pathogenesis of vascular remodeling. Loss of VSMC functions and activation of inflammation are thought to weaken the arterial wall and increase the risk of AAD formation and rupture [3][4][5][6][7].
Smooth muscle 22α (SM22α) is an actin-associated protein abundant in contractile VSMC and is widely used as a phenotypic marker to identify VSMC phenotypic transformation. SM22α decorates the contractile filament bundles within cultured VSMC exhibiting differentiated phenotypes. Decrease in expression of SM22α has been demonstrated in human atherosclerotic lesions, neointima formation [8,9], abdominal aortic aneurysm (AAA) [10,11], and thoracic aortic dissection [12], and mouse models [13,14]. Our previous studies demonstrate that the arteries of Sm22α -/mice develop enhanced inflammatory response and ROS production, which is involved in aortic aneurysm through different signaling mechanisms [15][16][17][18], suggesting that SM22α expression is closely associated with the structure and function of vascular media.
However, a report directly addressing circulating SM22α levels in AAD patients is still lacking. Thus, on the basis of such premises, we investigated serum SM22α level in patients with AAD compared with patients with only carotid artery stenosis (CAS) and normal controls, and assessed it value as diagnostic and monitoring markers in AAD.

Animals
Male C57BL/6J mice were obtained at 6~8 weeks of age from the Hebei Medical University Experimental Animal Center (Hebei, China). Mice were maintained under standard conditions at 22˚C with a 12-hour light/dark cycle and free access to food and water. For the induction of AAA, C57BL/6J mice were induced by perivascular application of CaPO 4 . A small piece of gauze soaked in 0.5M CaCl 2 was applied perivascularly for 15 min. This gauze is then replaced with another piece of phosphate buffered saline (PBS)-soaked gauze for 5 min. Aortic aneurysm rupture model of mice was infused with β-aminopropionitrile (BAPN 300 mg/kg/ d) combined with Ang II (4.0 mg/kg/d) simultaneously for 21 days via intraperitoneal injection. BAPN and Ang II were purchased from Sigma (St. Louis, MO). The neointimal formation of the C57BL/6J mice was induced by complete ligation of the left common carotid artery for 14~28 days. Ldlr −/− mice (at 6~8 weeks of age from Ex&Invivo Biotech Co.td, Hebei, China) were fed a high fat diet (HFD) containing 0.5% cholesterol and 20% fat for 12 weeks to induce atherosclerosis development. All animals were euthanized using intraperitoneal overdose anesthesia with sodium pentobarbital (200~250 mg/kg) at the end of the experiments, and the vascular tissues and blood samples were collected. All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, 8th Edition, 2011), and were approved by the Institutional Animal Care and Use Committee of Hebei Medical University.

Patients
The Medical Ethics Committee of Hebei Medical University approved all protocols using human samples (No. 2017042). All participants provided written informed consent prior to their participation in the study.
From May 2020 to March 2021, according to the computed tomography angiography results, clinical features, and diagnostic clinical results, a total of 41 AAD patients were admitted to the Vascular Surgery Department of the Fourth Affiliated Hospital of Hebei Medical University for their first-ever AAD. All of these people were treated via emergency surgical repair. May 2020 was chosen at the commencement time for this study and patients were selected on the basis of the following inclusion criteria: (1) emergency surgical treatment of Stanford type A dissection or type B dissection thoracic aneurysm; (2) no history of neoplasm or autoimmune, infectious, or inflammatory systemic diseases; (3) no presence of genetic syndromes known to be responsible for aortic disease. Serum samples were collected at 5 time points: preoperative, day 1, 5, 7 and 14 postoperative.
107 CAS patients were included to assess the difference in serum SM22α between AAD and atherosclerotic carotid stenosis disease in the same period. All patients were from the Department of Cardiovascular Medicine and underwent Duplex Carotid Ultrasound examination, which were enrolled on the basis of the following inclusion criteria: (1) when a plaque was identified in the carotid artery, the following degree of stenosis categorized by hemodynamic criteria were recorded: mild (<50%), moderate (50%~69%), and severe stenosis (�70%); (2) no cardiac causes of stroke; (3) no history of neoplasm or autoimmune or inflammatory systemic diseases; and (4) no familiar or personal history of AAD. 40 normal controls (NC group) matched for age and sex were included in this study to obtain reference values for serum levels of SM22α. These were selected on the basis of the following exclusion criteria: (1) no presence of genetic syndromes known to cause aortic disease; (2) no family history of AAD or atherosclerotic cardiovascular disease; (3) no history of AAD or atherosclerotic disease; (4) no diabetes mellitus; (5) no dyslipidaemia; and (6) no uncontrolled hypertension.
Since this was a retrospective observational study, the sample size was informed by the available participants during the study period rather than as a result of a prespecified sample size estimate.

Study variables and criteria
Clinical data including patient age, sex, smoking, drinking and blood pressure were recorded during clinical reviews or were obtained from previous hospital admission records. Smoking was defined as current smoking (smoking within the last month). Drinking was defined as an intake of more than one standard cup of Chinese liquor, one large bottle of regular beer, or one double measure of red wine at a time more than three times a week. Blood pressure was measured from each patient's upper right arm in a sedentary position using an automated sphygmomanometer after a 5-min rest. Hypertension was defined as an office blood pressure of 140/90 mmHg and above. All blood variables, including levels of fasting plasma glucose (FPG), serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c), were measured concomitantly. Abnormal blood glucose was diagnosed at FPG�7.0 mmol/L. Dyslipidemia was generally defined as the serum level of TC of 6.19 mmol/L and above, the serum level of TG of 2.27 mmol/L and above, the serum level of LDL-c of 4.14 mmol/L and above.

Measurement of SM22α level in serum
Serum levels of SM22α were measured using enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's instructions (cat.EK6896 & cat.EK6897, Signalway Antibody, USA). All serum samples should be tested initially without any dilution. Final SM22α serum levels were obtained based on the dilution of samples which corresponded to the linear portion of the standard curve.

Immunofluorescence analysis
Immunofluorescence staining was performed on 6-μm-thick frozen sections. Sections were blocked using 5% normal goat serum in TBS for 30 min and then incubated with primary antibodies against SM22α (Abcam, ab14106, 1:100) at 4˚C overnight, and isotype matched controls. Sections were washed 3 times with TBS and incubated with fluorescein-conjugated secondary antibodies (Alexa Fluor 1 543, Invitrogen) at a 1/200 dilution for 1 h at room temperature. Nuclei were detected by DAPI (Antifade Mountant with DAPI, Thermofisher). Images were acquired using a Confocal Laser Scanning Microscope Systems (Leica). Digitized images were analyzed with software program LAS AF Lite.

RNA isolation and quantitative reverse transcription-PCR (qRT -PCR)
Total RNAs from vascular tissues of mice were isolated using TRIzol reagent (Life Technologies). The nuclear and cytoplasmic fractions were extracted using Minute™ Cytoplasmic and Nuclear Extraction Kit (Invent Bio technologies). To quantify the amount of mRNA and cir-cRNA, cDNAs were synthesized using the M-MLV First Strand Kit (Life Technologies), and quantitative PCRs were performed using SYBR Green qPCR SuperMix-UDG (Life Technologies). For quantification, all RNA expression was normalized to the amount of Tubulin using the 2 -ΔΔCt method. For RT-PCR analysis, the following specific primers were used: SM22α forward, 5΄-CAACAAGGGTC CATCCTACGG-3΄ and reverse, 5΄-ATCTGGGCGGCCTACATCA-3΄; β-actin forward, 5΄-CGAGGCCCAGAGCAAGAGAGGTAT-3΄ and reverse, 5΄-CAC GGTTGGCCTTAGGGTTCA-3΄.

Statistics
Data analysis was performed with SPSS version 16.0. Data are presented as the means ± standard error of the mean (SEM), while categorical variables are presented as numbers or percentages. Differences between two groups were compared by t-tests. Associations were analyzed using Pearson correlations and linear regression models. The clinically important variables were selected for multiple linear regression analysis. Receiver operating characteristic (ROC) curve analysis was performed to assess the area under the curve (AUC) and Youden index was used to determine the best cut-off value of serum SM22α levels for predicting AAD in study subjects. Significance was taken at P<0.05 throughout, and denoted with one, two and three asterisks when lower than 0.05, 0.01 and 0.001, respectively.

Serum SM22α levels are correlated with vascular disease in mice
We showed that the incidence of AAA induced by CaPO 4 was 50% (4/8). Obviously, the maximum diameter of abdominal aorta was enlarged in mice induced by CaPO 4 (P<0.001, Fig 1A  and 1B), accompanied by increased elastin disruption and degradation ( Fig 1F). Immunofluorescence staining, qRT-PCR and Western blotting indicated that the expression of SM22α was remarkably decreased in AAA tissues compared with normal aortic tissues (P<0.001), and negatively correlated with the maximum diameter of abdominal aorta (r = -0.825, P<0.05, Fig  1G-1I). To validate VSMC phenotypic states in this study, we also detected other markers of phenotypic switching, including contractile VSMC markers (α-SMA) and synthetic VSMC marker (OPN). Expression of SM22α and α-SMA were significantly decreased while the expression of OPN was significantly increased in aortic aneurysm tissues compared to control aortic tissues. The phenotypic changes of VSMC are from constriction to synthesis, which can lead to change of tunica media character and eventually lead to aortic aneurysm. Aortic aneurysm rupture model of mice was established by administering β-aminopropionitrile (BAPN), a lysyl oxidase inhibitor, and angiotensin II (Ang II) to induce hypertension and degeneration of the elastic lamina, which would eventually result in the onset of aneurysm rupture. In this model, cotreatment with BAPN and Ang II caused an 85.7% (6/7) aneurysm incidence, with 71.4% (5/7) of these having aortic aneurysm rupture (Fig 1C).
Similarly, histomorphological and immunofluorescence staining showed that the expression of SM22α in aneurysm tissues was significantly decreased with the development of aneurysm ( Fig 1G).
As shown in Fig 1D and 1E, serum SM22α levels were decreased in AAA model of mice induced with CaPO 4 (P<0.001), which were consistent with reduction of SM22α levels in

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SM22α as a new biomarker for aortic aneurysm/dissection lesion tissues and negatively correlated with the rate of smooth muscle cells apoptosis in arterial media (r = -0.778, P<0.05, Fig 1J). Similar changes were found in a mouse model of BAPN and Ang II-induced aortic aneurysm formation and rupture. At an early stage of aneurysm development (after 2 weeks of co-administration of BAPN and Ang II), serum SM22α levels were markedly decreased compared with saline group (P<0.001), and significantly elevated in mice with ruptured aneurysm after 3 weeks administration (P<0.001), suggesting that it may be associated with SM22α released into the blood stream during the rupture process of aneurysm.
Expression of SM22α in tissues and serum specimens displays a similar profile in mice, which is also reflected in other arterial injury models in mice. During the neointimal formation induced by the carotid artery ligation, the expression of SM22α in the carotid artery ligation group was significantly decreased (Fig 2G-2I), accompanied with increased intima/media (I/M) area ratio (Fig 2C and 2D). SM22α expression was negatively correlated with I/M area ratio (r = -0.913, P<0.05). Similarly, the expression of SM22α in the athero-sclerotic plaque of HFD-fed mice was significantly decreased and negatively correlated with plaque size (r = -0.913, P<0.05, Fig 2E and 2F), suggesting that the expression of SM22α may reflect the structure and function of vascular media. A significant decrease in the serum levels of SM22α was observed in carotid artery ligation and HFD-fed models of mice compared with normal controls (P<0.001, Fig 2A and 2B), and decreased serum SM22α levels were associated with progression of vascular lesions (P<0.001).
We further explored whether the serum SM22α levels could be used to evaluate the severity of vascular media injury. The results showed that serum SM22α levels were negatively correlated with the I/M area ratio in neointimal formation (r = -0.874, P<0.001, Fig 2D) and plaque size of HFD-fed mice (r = -0.855, P<0.05, Fig 2E and 2F). Next, the carotid artery ultrasound results of HFD-fed mice showed that the systolic diameter and diastolic diameter were significantly decreased, the vessel wall thickness and the resistance index were significantly increased (Fig 2J). Correlation analysis showed that serum SM22α levels were negatively correlated with the vessel wall thickness and resistance index (r = -0.8745, r = -0.8714, P<0.001). These data indicated that SM22α serum levels closely paralleled the increasing degree of arterial injury and could be a potential serum marker for detecting the occurrence and development of arterial injury.

Serum level of SM22α in patients with AAD and CAS
Serum SM22α levels were investigated in 41 patients diagnosed with AAD undergoing surgery and compared with 107 CAS patients and 40 normal controls (Table 1).
In this study, we simultaneously detected serum SM22α levels in patients with AAD before and after surgical intervention. As shown in Fig 3A and 3B, the serum SM22α level of AAD patients (n = 41) at admission (3.080±0.370 ng/mL) was significantly higher than that of the normal controls (2.297±0.122 ng/mL, P<0.001). Next, we compared the serum SM22α levels between type A dissection patients (n = 34) and type B dissection thoracic aneurysm patients (n = 7), serum SM22α levels were significantly increased in patients with type A dissection and type B dissection thoracic aneurysm (3.189±0.300 ng/mL, 2.595±0.121 ng/mL, respectively, P<0.05) compared with the normal controls. More interestingly, we observed sharp differences in serum SM22α levels between these two groups (P<0.05), speculating that the serum SM22α levels may be related to the lacerated range and degree. Serum SM22α levels were markedly decreased at day 1 after surgery, and it was significantly reduced to the normal range on the 14th day after operation (2.190±0.230 ng/mL, P<0.05).
In addition, we measured serum SM22α levels in 107 patients with CAS. These patients were categorized into mild (n = 27), moderate (n = 40) and severe (n = 40) carotid stenosis groups by ultrasonography, we found that serum SM22α levels were dramatically lower in CAS patients (1.797±0.204 ng/mL, Fig 3C). Meanwhile, in the patients with CAS, the levels of serum SM22α also showed significant differences with increasing stenosis. The reduction of serum SM22α level was the most obvious in severe stenosis group (1.572±0.091 ng/mL) compared with that in mild stenosis group and moderate stenosis group (1.975±0.074 ng/mL, 1.902±0.128 ng/mL, respectively, P<0.0001, Fig 3C). The difference of serum SM22α levels between patients with AAD and CAS indicates that SM22α is specifically released into peripheral blood in patients with AAD.

Diagnostic value and correlation of serum SM22α levels with clinical parameters in patients with AAD and CAS
According to the ROC curve results, the AUC of serum SM22α for predicting AAD was 0.996, P<0.0001, sensitivity and specificity were 100% and 95%, respectively, the cut-off value was 2.514 ng/mL. When the levels of serum SM22α are greater than or equal to 2.514 ng/mL, the risk of AAD significantly increases (Fig 3E). These results suggest that the serum levels of SM22α have potential to be used as diagnostic biomarkers for AAD. We also found that the AUC value, sensitivity, and specificity of serum SM22α for predicting CAS was 0.977, 85% and 100%, respectively, the cut-off value was 2.028 ng/mL (Fig 3F), when the levels of serum SM22α are lower than or equal to 2.028 ng/mL, the risk of CAS significantly increases, suggesting that the detection of serum SM22α level may be used for rapid screening of the risk of CAS, with high specificity.
Correlation analysis showed that serum SM22α level in patients with AAD was particularly associated with FPG (r = 0.350, P<0.05), TC (r = 0.309, P<0.05), HDL-c (r = -0.372, actin. (I) Western blot for the expression of SM22α in mice aortas of each group (n = 3). β-actin served as a loading control. (J) Carotid ultrasonic examination in normal diet (ND) and HFD group, including representative images, systolic diameter, diastolic diameter, vessel wall thickness, resistance index. All data are expressed as mean±SEM. � P<0. 05

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SM22α as a new biomarker for aortic aneurysm/dissection P<0.05, Table 2), while further multiple linear regression analysis basically ruled out the interference of those related indicators on the serum SM22α level of AAD patients (P>0.05, Table 3).
In addition, as shown in Fig 3D and Table 2, serum SM22α levels in CAS patients were negatively correlated with the degree of carotid stenosis (r = -0.939, P<0.001), and there were also close correlations between serum SM22α and age (r = 0.329, P<0.001), DBP (r = -0.265, P = 0.003), TG (r = -0.283, P = 0.002), HDL-c (r = 0.290, P = 0.001) in patients with CAS. However, multiple linear regression analysis showed that only the degree of carotid artery stenosis was an independent related factor for serum SM22α level in patients with CAS (P<0.001, Table 4).

Discussion
Our studies and others have demonstrated that the disruption of SM22α impairs vascular structure and functions via promoting VSMC phenotype switching [7,14,19], whereas elevated SM22α expression also serves as a marker of VSMC senescence and hypertension [20]. Schellekens et al. found that plasma SM22α levels were closely paralleled the increasing degree of intestinal transmural damage upon progression of the duration of ischemia and a major part of the SM22α protein leaked out of the smooth muscle cells into blood [21]. However, this was not systemically studied by comparing serum, tissue and clinical data. So far, there is currently

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SM22α as a new biomarker for aortic aneurysm/dissection no specific serum biomarker of the media damage that might indicate VSMC phenotypic states and reflect the apoptosis of smooth muscle cells.
In animals, we demonstrate, for the first time, that SM22α serum levels are paralleled with its lower tissue expressions and may affect VSMC-specific contribution during the repair process after vascular injury, which drive damage of arterial media and reflect the progression of arterial media remodeling diseases. Importantly, the increased serum level of SM22α is associated with the occurrence of AAD. On the other hand, we confirmed that serum SM22α level in mice is negatively correlated with the rate of smooth muscle cells apoptosis in arterial media, the ratio of intima/media (I/M) area, atherosclerotic plaque size and extent. These data demonstrate that serum SM22α level as a specific biomarker in smooth muscle cells contributes to assess the structure and function of vascular media. Typically, serum biomarkers should be potentially used to screen patients with compatible symptoms. Smooth muscle myosin heavy chain (SM-MHC) is a major component of medial smooth muscle, which is also present in uterine and intestinal smooth muscle [22]. Previous studies showed that in patients with aortic dissection admitted to the hospital within 24 hours of onset, SM-MHC levels in serum were greatly raised. At 24 hours after operation, however, all cases showed a rapid decrease to normal values [23,24]. Similarly, we reviewed 41 patients with AAD to examine the value of SM22α in diagnosing AAD. The results suggested that the serum level of SM22α was significantly increased in patients with AAD at admission and showed potential diagnostic value for AAD. That is, if patients with unexplained chest pain have high serum SM22α levels at admission, they may be at high risk of AAD and should receive aggressive monitoring and therapeutic interventions. In addition, our result obtained from the comparison of SM22α levels between AAD and CAS patients seems to be of particular interest, we found that serum SM22α levels were significantly increased in AAD patients compared with CAS patients and health controls, suggesting that SM22α is specifically released in patients at high risk of AAD. We also found that serum SM22α levels were markedly decreased at day 1 after surgery, and it was significantly reduced to the normal range on the 14th day after operation, suggesting that dynamic changes of serum SM22α level are potentially useful for a better understanding of AAD disease course and progression, meanwhile, they provide serological basis for the early intervention.
The following serum markers, in contrast, have its limitations. Elastin is one of the main structural components of the arterial wall. As one of the main pathological features of the aortic media in aortic dissection is elastin lamellar disruption, elastin degradation products (sELAF) could potentially be released into the circulation at the time of aortic dissection, which may reflect the damage of vascular media [25]. Shinohara et al. indicated that high sELAF serum levels were directly associated with aortic dissection. But sELAF remain elevated for a period of 72 hours in patients with aortic dissection, therefore, it is not suitable for early screening [26].
Serum-based SM22α tests are potentially more acceptable for screening of the general and high-risk population, as they are high specificity and sensitivity. Although our biomarker model based on the serum SM22α levels showed high performance in distinguishing arterial injury patients from the controls, there were still some limitations to our study. First, baseline levels of serum SM22α in normal controls, and the elevation of serum SM22α in AAD patients should be further validated with a large number of patients. Second, the causality of SM22α and other additional markers, especially those relating to inflammation and endothelial function, could not be inferred, the simultaneous measurement of other additional markers might expand our understanding. Third, we did not detect dynamic changes in plasma SM22α levels in the AAD patients. Finally, association between serum SM22α levels with in-hospital death needs to be confirmed in a large-scale study, as we had no death events in 41 AAD patients. We did not follow-up patients to assess long-term mortality or prognosis, either. Thus, we need to conduct follow-up studies to detect the clinical outcomes of these patients.
In conclusion, this study shows that serum SM22α level could serve as a potential biomarker for discerning AAD patients from CAS patients and the healthy population.
Supporting information S1